Arabidopsis BTB-A2s Play a Key Role in Drought Stress

Simple Summary Given that drought stress may particularly threaten plant survival and crop yields, plants have developed sophisticated adaptive strategies including drought tolerance, escape, and avoidance strategies, for dealing with drought stress. Abscisic acid has been widely acknowledged as a principal signaling molecule in plants responding to drought, reducing water loss by prompting stomatal closure while activating various stress-responsive genes. Broad-complex, Tramtrack, and Bric-à-brac (BTB) proteins in plants are important for plant growth and stress responses. The Arabidopsis btb-a2.1/2/3 mutant confers drought tolerance by modulating plant growth parameters, physiology, and gene expression. Overall, AtBTB-A2s negatively regulate drought tolerance by suppressing stomatal closure and weakening ABA signaling. The results revealed the new physiological activity of AtBTB-A2s within Arabidopsis and the possible mechanism that mediates ABA-dependent signaling pathways during drought stress. Abstract Drought stress significantly impacts plant growth, productivity, and yield, necessitating a swift fine-tuning of pathways for adaptation to harsh environmental conditions. This study explored the effects of Arabidopsis BTB-A2.1, BTB-A2.2, and BTB-A2.3, distinguished by their exclusive possession of the Broad-complex, Tramtrack, and Bric-à-brac (BTB) domain, on the negative regulation of drought stress mediated by abscisic acid (ABA) signaling. Promoter analysis revealed the presence of numerous ABA-responsive and drought stress-related cis-acting elements within the promoters of AtBTB-A2.1, AtBTB-A2.2, and AtBTB-A2.3. The AtBTB-A2.1, AtBTB-A2.2, and AtBTB-A2.3 transcript abundances increased under drought and ABA induction according to qRT-PCR and GUS staining. Furthermore, the Arabidopsis btb-a2.1/2/3 triple mutant exhibited enhanced drought tolerance, supporting the findings from the overexpression studies. Additionally, we detected a decrease in the stomatal aperture and water loss rate of the Arabidopsis btb-a2.1/2/3 mutant, suggesting the involvement of these genes in repressing stomatal closure. Importantly, the ABA signaling-responsive gene levels within Arabidopsis btb-a2.1/2/3 significantly increased compared with those in the wild type (WT) under drought stress. Based on such findings, Arabidopsis BTB-A2s negatively regulate drought stress via the ABA signaling pathway.


Introduction
Plants with a sessile life cycle must adapt to their surroundings to survive, facilitating individual growth and population reproduction.They often encounter various environmental challenges, such as extreme temperatures, salinity, drought, and pathogens, which can significantly affect crop productivity and quality.Among these challenges, drought poses a particularly critical threat, impacting the ability of plants to thrive on land [1].Throughout the evolution of land plants, sophisticated adaptive strategies have emerged to cope with these environmental pressures.Specifically, in response to drought, plants have developed three main strategies: drought tolerance, escape, and avoidance [2][3][4].Drought tolerance involves the maintenance of growth using a limited water content during drought Biology 2024, 13, 561 2 of 14 via mechanisms such as scavenging reactive oxygen species (ROS), adjusting osmotic balance, and activating stress-related genes.Drought escape entails accelerating the plant's life cycle to complete reproduction before stress becomes too severe.Drought avoidance focuses on minimizing water loss by quickly closing the stomata and delaying growth until favorable conditions return [2,4,5].The most effective way among these adaptive measures is stomatal closure, which is achieved by curtailing water loss in a water-deficit environment [3,6,7].
Recent research advances have highlighted the significant impact of abscisic acid (ABA), a pivotal plant hormone and stress regulator, on regulating plant growth and adapting to stressors.Its effects include various crucial functions, including stomatal closure, seed germination, responses to drought and cold, and defense against pathogens [8].ABA significantly affects orchestrating stomatal closure, particularly upon drought conditions [9].More specifically, drought stress rapidly promotes stomatal closure in a manner predominantly dependent on ABA.Research has explored the stimulation of ABA production and accumulation within guard cells upon water deficit signals.Additionally, active synthesis occurs in other tissues, such as roots and leaf vasculature, facilitating transport to guard cells [9][10][11].ABA accumulation can be recognized by specific receptors known as pyrabactin resistance (PYR)/PYR1-like (PYL)/regulatory components of ABA receptor (RCAR) receptors.These receptors deactivate clade A type 2C phosphatases (PP2C), subsequently activating downstream SNF1-related protein kinase 2.2 (SnRK2.2),SnRK2.3, and SnRK2.6 [12][13][14].Among those members of the module, SnRK2s are important for positively accommodating ABA signal transduction to turn ABA signals on or off.Activated SnRK2s can then regulate stomatal closure to cope with water deficit by activating and phosphorylating channel proteins like SLAC1, KAT1, and numerous transcription factors such as ABI5, ABF2/AREB1, ABF3, and ABF4/AREB2 [15][16][17][18].
Broad-complex, Tramtrack, and Bric-à-brac/poxvirus and zinc finger (BTB/POZ) proteins, distinguished by approximately 120 conserved residues at their N-terminus, also referred to as the BTB domain, have been extensively investigated in eukaryotes [19,20].Typically, these proteins act as potential substrate adaptors for CUL3 through the BTB domain and for substrate proteins via another protein-protein interaction domain [19].Exploring the gene family of BTB domain-containing proteins across diverse plant varieties, including Arabidopsis, tomato, rice, corn, peach, and cucumber, can highlight their diverse biological functions [20][21][22][23][24][25].In various biological processes, including chromatin organization, transcriptional regulation, cytoskeletal modulation, and protein degradation, BTB domain-containing proteins predominantly influence plant growth and environmental stress responses.Recent investigations have revealed their significance in plant stress responses.For instance, in Arabidopsis, non-expressers of pathogenesis-related genes NPR1, NPR3, and NPR4, which are salicylic acid receptors, have all been recognized as BTB/POZ proteins, exerting contrasting effects on the transcriptional control of the salicylic acidmediated expression of defense genes [20,26].Moreover, a tobacco BTB/POZ domain E3 ligase protein, POB1, restrains hypersensitive response programmed cell death (HR-PCD) in plant innate immune responses by facilitating the ubiquitin degradation of PUB17 [27].In cucumber, salt stress markedly decreases the expression of the CsBT1 gene [25].Moreover, the sweet potato IbBT4 gene confers drought tolerance [28].OsMBTB32 enhances rice growth upon salt stress by interacting with OsCUL1s [29].
Previously, our research revealed that AtBTB-A2 proteins, expressed within the guard cell cytoplasm and nucleus, can interact with SnRK2.3 while influencing its stability, thereby impacting seed germination.Additionally, they also interact with SnRK2.6 [30].According to the above results, it was hypothesized that AtBTB-A2 proteins were probably important for regulating responses to drought stress, a significant environmental challenge for plants.
To explore this hypothesis further, we investigated the biological functions and mechanisms of AtBTB-A2 proteins associated with drought stress.Our results indicate that ABA and drought stress alter the transcript abundances of AtBTB-A2.1,AtBTB-A2.2,and AtBTB-A2.3.The genetic data suggest that AtBTB-A2 proteins function in drought stress responses, thereby influencing ABA signaling responsive gene expression in the meantime of modulating drought tolerance.Therefore, our findings suggest that the negative regulatory roles of AtBTB-A2s in ABA responses lead to reduced drought stress tolerance, providing insights into their function within the intricate network of drought stress adaptation.

Plant Materials and Growth Conditions
The plants used were generated in an Arabidopsis Columbia-0 (Col-0) background.The generation and identification of btb-a2.1/2/3triple mutant and AtBTB-A2 overexpression lines have been detailed previously [30].Transgenic plants were cultivated to the homozygous (T3) generation through selection with 10 µg/mL BASTA (Sigma-Aldrich, Milwaukee, WI, USA).
For the plant propagation and growth experiments, 75% ethanol was added to sterilize the surface of the Arabidopsis seeds for 5 min, followed by rinsing thrice using sterile water before sowing on half-strength Murashige and Skoog (MS) Phytoagar media that contained 1% (w/v) sucrose (pH 5.8).Following stratification for 3 days under 4 • C, we transferred the seeds to a growth incubator to achieve germination and growth with 16 h/8 h light/dark conditions at 22 • C. In soil culture, we transferred 10-day-old plants into nutrient-rich soil (Pindstrup Mosebrug, Denmark) for growth within the greenhouse at a light intensity of 150 µmol/m 2 /s, a temperature of 22 • C, and a light/dark cycle of 16 h/8 h.

Drought Assay
To ensure consistent soil moisture, each pot, filled with an equal amount of soil, was saturated with water.Sixteen 7-day-old plants displaying uniform growth, were planted in each small pot under identical humidity conditions.The control seedlings received watering every 3 days, while experimental seedlings underwent gradual drought stress through halting watering until the drought phenotype emerged, which was then followed by a 3-day period of watering.

Water Loss Determination
In the water loss assays, we detached the entire rosette from independent 4-week-old seedlings of each sample and placed them on Whatman filter paper on a laboratory bench (21)(22) • C, about 40% relative humidity).Leaf fresh weight was measured and recorded at the indicated points, and fresh weight loss was analyzed.The test was replicated three times.

MDA Content Determination
We determined the MDA content in accordance with the method proposed by Kong [32].One hundred milligrams of Arabidopsis leaves from normal or drought-stressed plants were ground in a cold mortar containing 0.1% (w/v) trichloroacetic acid (2 mL, TCA) as well as 8 mL of TCA for further grinding.After 10 min of centrifugation of the homogenate at 4000× g and 4 • C, 0.6% thiobarbituric acid (TBA) reagent (2 mL) was added to the supernatants (2 mL, with an equivalent amount of distilled water as a control).After a 15 min reaction in a boiling water bath, the reaction system was rapidly cooled, and centrifugated for a 10 min period at 5000× g.Supernatants were collected to measure the absorbance at wavelengths of 532 nm, 600 nm, and 450 nm.MDA contents were determined by the 155 mM −1 cm −1 extinction coefficient as described previously [33].

Relative Electric Conductivity (REC) Determination
The REC was measured according to the method proposed by Kong [32].Briefly, the Arabidopsis leaves were rinsed with deionized water twice, and later, the surface moisture was removed using clean filter paper.Thirty leaf discs from each line were distributed into three clean tubes with ten discs in each tube.Deionized water (10 mL) was introduced into each tube containing leaf discs, which were then vacuumed for 20 min to remove air from the intercellular spaces and subsequently shocked for 1 h to measure the initial conductivity (S1) using a conductivity meter.Each tube was positioned for a 10 min period within the boiling water bath and later cooled to room temperature.The materials were equilibrated at ambient temperature for a 10 min period and shaken well for measuring final conductivity (S2).We recorded distilled water conductivity (S0) as a blank control.The REC could be determined as REC = (S1 − S0)/(S2 − S0) × 100.

Physiological Measurements of Guard Cells
The stomatal aperture test was carried out in line with Eisele's method [34].We first obtained the fourth expanded rosette leaves from one-month-old Arabidopsis plants.Following light incubation for a 2 h duration with stomatal opening buffer (10 mM MES, 10 mM KCl, 0.1 mM CaCl 2 , and pH 6.15) with the abaxial leaf surface downward to make the stomata as wide open as possible, the leaves were incubated within stomatal opening buffer that contained 10 µM ABA (Sigma-Aldrich, Milwaukee, WI, USA) for the indicated time points, and the epidermis on the back of the leaves was quickly peeled off.The stomata were observed under a microscope.ImageJ was utilized to determine the stromal width and length, while stomatal aperture indices were determined as the specific value of width compared with length.

RNA Isolation and qRT-PCR
RNA extraction was completed from Arabidopsis plants using TRIzol reagent (Invitrogen).First-strand cDNA was prepared with M-MLV reverse transcriptase (Promega, Madison, WI, USA) for RT-PCR with gene-specific primers.Thereafter, the Bio-Rad C1000 Thermal Cycler system was used for qRT-PCR with a SYBR Green I Master kit (Roche Diagnostics, Mannheim, Germany) for analyzing AtBTB-A2s expression within Arabidopsis seedlings under drought stress conditions and for examining ABA synthesis-related and ABA-responsive gene levels.AtACTIN2 (AT3G18780) served as an internal control gene.The data were acquired from 3 biological replicates.Table S4 displays the primers used.

Statistical Analysis
All results in the current work were acquired from 3 separate experiments.The statistical analysis was conducted using Student's t-test (* p < 0.05, ** p < 0.01).
Meanwhile, the btb-a2.1/2/3-treatedleaves subjected to the isolated leaf assays in 15% PEG-6000 solution showed less severe purple discoloration than did the wild-type leaves
Meanwhile, the btb-a2.1/2/3-treatedleaves subjected to the isolated leaf assays in 15% PEG-6000 solution showed less severe purple discoloration than did the wild-type leaves after 12 h.Trypan blue staining is used as an indicator of cell death.The leaves from the wild-type plants had deeper staining compared with those from the btb-a2.1/2/3mutant Biology 2024, 13, 561 6 of 14 plants, suggesting a greater cell death degree within wild-type leaves than mutant leaves (Figure 2c).
Under stress conditions such as drought, cold, or salt stress, the relative electrical conductivity (REC) increases due to a reduction in membrane integrity, and the malondialdehyde (MDA) content increases because of membrane system damage [35].Therefore, we measured the MDA content and REC, which serve as important indicators for evaluating drought resistance in plants.The MDA content and the REC did not show notable differences in the wild type compared with mutant plants under normal watering conditions.Although the MDA content and REC were elevated within the water-deficit treatment group, these indicators were significantly lower in the btb-a2.1/2/3treatment group compared with the wild-type group (Figure 2d,e).Based on the above findings, the Arabidopsis btb-a2.1/2/3mutant enhances drought resistance.
Biology 2024, 13, x FOR PEER REVIEW 6 of 14 after 12 h.Trypan blue staining is used as an indicator of cell death.The leaves from the wild-type plants had deeper staining compared with those from the btb-a2.1/2/3mutant plants, suggesting a greater cell death degree within wild-type leaves than mutant leaves (Figure 2c).Under stress conditions such as drought, cold, or salt stress, the relative electrical conductivity (REC) increases due to a reduction in membrane integrity, and the malondialdehyde (MDA) content increases because of membrane system damage [35].Therefore, we measured the MDA content and REC, which serve as important indicators for evaluating drought resistance in plants.The MDA content and the REC did not show notable differences in the wild type compared with mutant plants under normal watering conditions.Although the MDA content and REC were elevated within the water-deficit treatment group, these indicators were significantly lower in the btb-a2.1/2/3treatment group compared with the wild-type group (Figure 2d, e).Based on the above findings, the Arabidopsis btb-a2.1/2/3mutant enhances drought resistance.

AtBTB-A2s Expression after ABA Treatment
In plants, ABA is a pivotal hormone that orchestrates many physiological responses under stress conditions, notably extreme temperatures, salinity, and drought [36].Previous investigations have indicated that AtBTB-A2s are negative modulators of ABA signal transduction in the seed germination phase in Arabidopsis [30].Thus, we delved into

Arabidopsis btb-a2.1/2/3 Reduced Water Loss under Drought Stress and Regulated ABA-Mediated Stomatal Closure
Evolution has equipped plants with strategies to combat drought stress, such as minimizing water loss to maintain limited water.Therefore, we assessed the water content upon drought stress.The wild-type plants had decreased relative water content relative to triple mutant btb-a2.1/2/3,indicating a more rapid water loss rate in the wild type compared with triple mutant btb-a2.1/2/3(Figure 4a).According to these results, the btb-a2.1/2/3mutant might confer drought tolerance by reducing the transpiration rate.
Plants reduce transpiration by closing their stomata, allowing them to survive under drought conditions.ABA facilitates stomatal closure or impedes stomatal opening, consequently minimizing water loss in aerial plant tissues [37].We examined the dynamics of stomatal opening and closing after ABA treatment.According to our results, the stomata of the mutant plants closed at an increased rate after ABA treatment relative to those of the WT plants (Figure 4b, c).Consequently, we speculate that differences in leaf water loss rates may be the main cause of differences in drought tolerance, while btb-a2.1/2/3may exert their effects by promoting stomatal closure upon drought stress.Evolution has equipped plants with strategies to combat drought stress, such as minimizing water loss to maintain limited water.Therefore, we assessed the water content upon drought stress.The wild-type plants had decreased relative water content relative to triple mutant btb-a2.1/2/3,indicating a more rapid water loss rate in the wild type compared with triple mutant btb-a2.1/2/3(Figure 4a).According to these results, the btb-a2.1/2/3mutant might confer drought tolerance by reducing the transpiration rate.
Plants reduce transpiration by closing their stomata, allowing them to survive under drought conditions.ABA facilitates stomatal closure or impedes stomatal opening, consequently minimizing water loss in aerial plant tissues [37].We examined the dynamics of stomatal opening and closing after ABA treatment.According to our results, the stomata of the mutant plants closed at an increased rate after ABA treatment relative to those of the WT plants (Figure 4b,c).Consequently, we speculate that differences in leaf water loss rates may be the main cause of differences in drought tolerance, while btb-a2.1/2/3may exert their effects by promoting stomatal closure upon drought stress.

Sensitivity of AtBTB-A2 Overexpression Lines to Drought Stress
Given the enhanced drought resistance observed from the btb-a2.1/2/3mutant, we investigated the drought response of AtBTB-A2.1,AtBTB-A2.2,and AtBTB-A2.3overexpression lines.As shown in Figure 5a, under normal conditions, wild-type plants showed similar growth to overexpression plants.Upon drought conditions, the overexpression lines of AtBTB-A2.1,AtBTB-A2.2,and AtBTB-A2.3exhibited pronounced wilting and decay, different from the healthier growth observed from wild-type plants.Following a 3day recovery period, most of the overexpression plants showed minimal recovery, while the wild-type plants recovered better.According to survival statistics, the survival rate of the wild type was around 64.6%, whereas the survival rates of the AtBTB-A2.1,AtBTB-A2.2,and AtBTB-A2.3overexpression lines were about 41.7%, 25%, and 20.8%, respectively (Figure 5b).Consistently, detached leaf assays further confirmed more rapid water loss in overexpression plants than in wild-type plants, resulting in earlier wilting (Figure 5c).Finally, an analysis of stomatal apertures revealed larger openings among the overexpression plants relative to the WT plants after ABA treatment (Figure 5d, e).Thus, the overexpression of AtBTB-A2.1,AtBTB-A2.2,and AtBTB-A2.3enhanced drought sensitivity through increasing stomatal aperture.

Sensitivity of AtBTB-A2 Overexpression Lines to Drought Stress
Given the enhanced drought resistance observed from the btb-a2.1/2/3mutant, we investigated the drought response of AtBTB-A2.1,AtBTB-A2.2,and AtBTB-A2.3overexpression lines.As shown in Figure 5a, under normal conditions, wild-type plants showed similar growth to overexpression plants.Upon drought conditions, the overexpression lines of AtBTB-A2.1,AtBTB-A2.2,and AtBTB-A2.3exhibited pronounced wilting and decay, different from the healthier growth observed from wild-type plants.Following a 3-day recovery period, most of the overexpression plants showed minimal recovery, while the wild-type plants recovered better.According to survival statistics, the survival rate of the wild type was around 64.6%, whereas the survival rates of the AtBTB-A2.1,AtBTB-A2.2,and AtBTB-A2.3overexpression lines were about 41.7%, 25%, and 20.8%, respectively (Figure 5b).Consistently, detached leaf assays further confirmed more rapid water loss in overexpression plants than in wild-type plants, resulting in earlier wilting (Figure 5c).Finally, an analysis of stomatal apertures revealed larger openings among the overexpression plants relative to the WT plants after ABA treatment (Figure 5d,e).Thus, the overexpression of AtBTB-A2.1,AtBTB-A2.2,and AtBTB-A2.3enhanced drought sensitivity through increasing stomatal aperture.

AtBTB-A2s Are Likely Related to Drought Stress Dependent on ABA Signaling Pathways
Previous research has demonstrated that AtBTB-A2s interact with SnRK2.6, which is important for the drought stress response [30].As the btb-a2.1/2/3mutant exhibited drought resistance, this prompted us to investigate the involvement of AtBTB-A2s during ABA-mediated drought stress responses.Initially, it was proposed that the NCED3 gene, which encodes critical and rate-limiting enzymes related to ABA biosynthesis, could regulate water stress through ABA accumulation [38].The AtBTB-A2.1,AtBTB-A2.2,and

AtBTB-A2s Are Likely Related to Drought Stress Dependent on ABA Signaling Pathways
Previous research has demonstrated that AtBTB-A2s interact with SnRK2.6, which is important for the drought stress response [30].As the btb-a2.1/2/3mutant exhibited drought resistance, this prompted us to investigate the involvement of AtBTB-A2s during ABAmediated drought stress responses.Initially, it was proposed that the NCED3 gene, which encodes critical and rate-limiting enzymes related to ABA biosynthesis, could regulate water stress through ABA accumulation [38].The AtBTB-A2.1,AtBTB-A2.2,and AtBTB-A2.3expression was analyzed in the wild type and the atnced3 mutant, revealing no significant difference between them (Figure S1a).Subsequently, we examined the ABA biosynthesisrelated gene (AtAAO3, AtABA1, AtABA3, and AtNCED3) expression levels in the wild type and the btb-a2.1/2/3mutant before and after exposure to drought conditions.Although drought stress increased the gene levels within both WT and mutant plants, no notable differences were detected (Figure S1b), implying that the expression of AtBTB-A2s may be not related to changes in endogenous ABA expression during drought stress.Finally, we detected the levels of ABA signaling-responsive genes (AtABI5, AtRAB18, AtRD29A, and AtRD9B) through qRT-PCR.We discovered that after drought stress, the levels of ABA signaling-responsive genes markedly increased, but the up-regulated expression was more significant in the btb-a2.1/2/3mutant compared with the WT (Figure 6).According to the above findings, AtBTB-A2s might exert a negative regulation on the drought stress response in a manner dependent on the ABA signaling pathway.
Biology 2024, 13, x FOR PEER REVIEW 10 of 14 AtBTB-A2.3 expression was analyzed in the wild type and the atnced3 mutant, revealing no significant difference between them (Figure S1a).Subsequently, we examined the ABA biosynthesis-related gene (AtAAO3, AtABA1, AtABA3, and AtNCED3) expression levels in the wild type and the btb-a2.1/2/3mutant before and after exposure to drought conditions.Although drought stress increased the gene levels within both WT and mutant plants, no notable differences were detected (Figure S1b), implying that the expression of AtBTB-A2s may be not related to changes in endogenous ABA expression during drought stress.Finally, we detected the levels of ABA signaling-responsive genes (AtABI5, AtRAB18, AtRD29A, and AtRD9B) through qRT-PCR.We discovered that after drought stress, the levels of ABA signaling-responsive genes markedly increased, but the up-regulated expression was more significant in the btb-a2.1/2/3mutant compared with the WT (Figure 6).According to the above findings, AtBTB-A2s might exert a negative regulation on the drought stress response in a manner dependent on the ABA signaling pathway.

Discussion
Upon environmental stresses, dramatic changes in physiology, metabolism, and especially gene expression often occur [39,40].In plants, BTB proteins exert vital effects on plant growth as well as stress responses.AtSIBP1, the potential substrate receptor for the CRL3 complex, positively modulates the salt stress response by suppressing ROS accumulation [41].MdBT2 can negatively regulate drought stress response by enhancing the transcription factor MdNAC143 degradation in apples [42].AtBPH1, a novel substrate receptor of CRL3, can exert a negative regulation of ABA-induced cellular responses, like drought stress and seed germination [43].Nonetheless, the exact effects of numerous BTB genes in plants, particularly stress responses, remain unknown.This study discovered that three Arabidopsis BTB-A2 subfamily genes negatively regulate drought resistance by suppressing stomatal closure and weakening ABA signaling.This research revealed the

Discussion
Upon environmental stresses, dramatic changes in physiology, metabolism, and especially gene expression often occur [39,40].In plants, BTB proteins exert vital effects on plant growth as well as stress responses.AtSIBP1, the potential substrate receptor for the CRL3 complex, positively modulates the salt stress response by suppressing ROS accumulation [41].MdBT2 can negatively regulate drought stress response by enhancing the transcription factor MdNAC143 degradation in apples [42].AtBPH1, a novel substrate receptor of CRL3, can exert a negative regulation of ABA-induced cellular responses, like drought stress and seed germination [43].Nonetheless, the exact effects of numerous BTB genes in plants, particularly stress responses, remain unknown.This study discovered that three Arabidopsis BTB-A2 subfamily genes negatively regulate drought resistance by suppressing stomatal closure and weakening ABA signaling.This research revealed the new physiological activity of AtBTB-A2s within Arabidopsis and revealed the possible mechanism that mediates ABA-dependent signaling pathways during drought stress.
Here, AtBTB-A2s transcript levels rapidly increased upon drought stress, suggesting that AtBTB-A2s may have the potential to function under drought stress.Considering that we previously reported that AtBTB-A2.1,AtBTB-A2.2,and AtBTB-A2.3are similar in terms of their localization and expression patterns and that the resulting polymers redundantly participate in biological activities [30], we constructed triple mutant and AtBTB-A2s overexpressing transgenic plants harboring these three proteins and screened their response to drought stress.According to the results, compared with the WT plants, the btb-a2.1/2/3mutant plants at the adult stage exhibited increased tolerance to drought stress (Figure 2a,b).Furthermore, this conclusion was supported by the results showing that btb-a2.1/2/3grew better than the wild type under PEG treatment, and the REL, MDA content, and degree of cell death in btb-a2.1/2/3decreased relative to those in the WT (Figure 2c-e).Conversely, the overexpression transgenic plants exhibited increased sensitivity to drought stress relative to the wild-type plants (Figure 5).Based on the above results, AtBTB-A2s may negatively regulate tolerance to drought stress.
Plants react to water-deficit stress via intricate signaling pathways, resulting in stomatal closure [44].ABA is widely acknowledged as the principal signaling molecule in the drought response of plants, reducing water loss by prompting stomatal closure and activating various stress-responsive genes [45,46].Initially, we observed a robust induction of AtBTB-A2s by external ABA according to GUS staining (Figure 3).Additionally, AtBTB-A2.1,AtBTB-A2.2,and AtBTB-A2.3interact with a component in the ABA signaling pathway, SnRK2.6 [30].Third, AtBTB-A2s inhibited stomatal closure via the ABA-dependent mode.Fourth, the btb-a2.1/2/3mutant impacted ABA-responsive gene expression, but not ABA synthesis-related gene levels under drought stress (Figure 6).These results confirm the ability of the btb-a2.1/2/3mutant to reduce plant water loss rate upon drought stress (Figure 4).According to the above findings, AtBTB-A2s confer sensitivity to drought stress by reducing water retention via the ABA-dependent signaling pathway.
Multiple plant BTB protein family members, containing the conserved BTB motif, can regulate substrate recognition while acting as substrate receptors for the Cul3 E3 ubiquitin ligase complex [47].Our data demonstrated that Arabidopsis BTB-A2s negatively affected the response of plants to drought stress via the ABA signaling-dependent manner.Moreover, several ABA signaling transducers are regulated via ubiquitination through their impact on protein localization, activity, interaction ability, and assembly, resulting in changes in ABA-mediated cellular events [48].We succeeded in identifying the potential target protein of AtBTB-A2s, SnRK2.6, which is also called open stomata 1 (OST1) and is a serine/threonine protein kinase with important effects on connecting ABA receptor complexes with downstream components, including responsive factors (RD29A, RD29B, RAB18, ABI5, and so on) and anion channels, thus regulating stomatal aperture in the stress response.Previous studies have indicated that the up-regulated transcript abundances of stress-responsive genes indicate that plants can manage drought stress [49].Additionally, under drought stress, the up-regulation of certain responsive factors, like the ABA signaling-related genes ABI5, RD29A, and RD29B, positively confer drought tolerance to plants [50][51][52][53].Some stress-responsive players have been discovered, but the mechanisms that regulate pathway activation duration and amplitude, which have a critical effect on stress adaptation, are largely unclear.Overall, our findings suggest that AtBTB-A2s likely participate in negatively regulating ABA signaling.ABA up-regulated AtBTB-A2 expression, thereby regulating SnRK2.6 during drought stress (Figure 7).However, how AtBTB-A2s affect SnRK2.6 upon drought stress needs further investigation to clarify the underlying mechanisms involved.

Conclusions
This study used reverse genetics methods to reveal a novel biological role and potential mechanism of Arabidopsis BTB-A2 genes in drought tolerance.According to the results, AtBTB-A2s exert a negative effect on the drought stress response.The present work sheds novel light on the physiological effects of BTB-A2s in Arabidopsis and offers further insight into the molecular mechanisms involved in ABA-dependent signaling pathways during drought stress.

Figure 4 .
Figure 4. Water loss of the btb-a2.1/2/3mutant decreased upon drought stress through ABA-induced stomatal closure.(a) Relative water content measured in detached leaves of control and mutant plants.(b) Stomatal opening of the WT and triple mutant plants at different time periods with and without 10 µM ABA.(c) Stomatal aperture measurements of the WT and triple mutant plants at varying time periods after a 10 µM ABA treatment.Approximately 100 stomas were analyzed for each line at each time point.The results are shown as mean ± SD from 3 biological replicates.* p < 0.05 and ** p < 0.01 stood for statistical significance in the wild type compared with btb-a2.1/2/3plants.

Figure 4 .
Figure 4. Water loss of the btb-a2.1/2/3mutant decreased upon drought stress through ABA-induced stomatal closure.(a) Relative water content measured in detached leaves of control and mutant plants.(b) Stomatal opening of the WT and triple mutant plants at different time periods with and without 10 µM ABA.(c) Stomatal aperture measurements of the WT and triple mutant plants at varying time periods after a 10 µM ABA treatment.Approximately 100 stomas were analyzed for each line at each time point.The results are shown as mean ± SD from 3 biological replicates.* p < 0.05 and ** p < 0.01 stood for statistical significance in the wild type compared with btb-a2.1/2/3plants.

Figure 5 .
Figure 5. AtBTB-A2.1,AtBTB-A2.2,and AtBTB-A2.3overexpressing plants exhibited enhanced drought sensitivity.(a) WT and overexpression transgenic plant phenotypes upon normal and drought stress conditions for 3-week days and after 3-day rewatering.(b) Survival ratio assay.(c) Measurement of relative water content by detached leaf samples in control and transgenic plants under drought conditions.(d) Stomatal opening of control and transgenic plants at specific time periods with/without 10 µM ABA.(e) Stomatal aperture measurements of control and transgenic plants at specific time periods under 10 µM ABA treatment.Approximately 100 stomas were analyzed for each line at each time point.The results are indicated by mean ± SD from 3 biological replicates.* p < 0.05 and ** p < 0.01 stood for statistical significance in the wild type compared with btb-a2.1/2/3plants.

Figure 5 .
Figure 5. AtBTB-A2.1,AtBTB-A2.2,and AtBTB-A2.3overexpressing plants exhibited enhanced drought sensitivity.(a) WT and overexpression transgenic plant phenotypes upon normal and drought stress conditions for 3-week days and after 3-day rewatering.(b) Survival ratio assay.(c) Measurement of relative water content by detached leaf samples in control and transgenic plants under drought conditions.(d) Stomatal opening of control and transgenic plants at specific time periods with/without 10 µM ABA.(e) Stomatal aperture measurements of control and transgenic plants at specific time periods under 10 µM ABA treatment.Approximately 100 stomas were analyzed for each line at each time point.The results are indicated by mean ± SD from 3 biological replicates.* p < 0.05 and ** p < 0.01 stood for statistical significance in the wild type compared with btb-a2.1/2/3plants.

Figure 6 .
Figure 6.qRT-PCR was carried out to measure ABA signal-responsive gene levels upon drought stress.(a-d) The expression levels of AtABI5, AtRAB18, AtRD29A, and AtRD29D in the WT and btb-a2.1/2/3mutant upon normal and drought conditions.The AtACTIN2 gene served as the endogenous reference.The results are presented as mean ± SD from 3 biological replicates.* p < 0.05 and ** p < 0.01 stood for statistical significance in the wild type compared with btb-a2.1/2/3plants.

Figure 6 .
Figure 6.qRT-PCR was carried out to measure ABA signal-responsive gene levels upon drought stress.(a-d) The expression levels of AtABI5, AtRAB18, AtRD29A, and AtRD29D in the WT and btb-a2.1/2/3mutant upon normal and drought conditions.The AtACTIN2 gene served as the endogenous reference.The results are presented as mean ± SD from 3 biological replicates.* p < 0.05 and ** p < 0.01 stood for statistical significance in the wild type compared with btb-a2.1/2/3plants.

Figure 7 .
Figure 7. Proposed model for negative effects of AtBTB-A2s under drought stress.

Author Contributions:
Conceptualization, G.C.; formal analysis, investigation, Y.Z., Z.W. and S.L.; funding acquisition, G.C.; supervision, G.C.; writing-original draft, G.C.; writing-review and editing, G.C. and G.W. All authors have read and agreed to the published version of the manuscript.Funding: The present work was funded by grants from the Natural Science Foundation of Shandong (ZR2022QC240), the Natural Science Foundation of Rizhao (RZ2021ZR19), and the Doctor Startup Foundation of Jining Medical University (600940001).Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.